The First Appearance of Specific Antigens during
the Induction of the Lens1
by
JAN
LANGMAN2
From the Department of Anatomy, McGill University, Montreal
INTRODUCTION
T H E formation of the lens in the chick embryo is known to depend upon 'inductive' influences from the eye-cup (Alexander, 1937; Van Deth, 1940; Waddington
& Cohen, 1936). A period of direct contact between eye-cup and presumptive
lens ectoderm from the 9- to the 20-somite stage is essential for the induction
(Weiss, 1947; McKeehan, 1951; Langman, 1956). At the beginning of this period
(9-12-somite stage), the cytoplasm of the presumptive lens ectoderm cells is
vacuolated and the nuclei have a random distribution, as in the ectodermal
epithelium of the head region. During subsequent development (13-16-somite
stage) the intracellular vacuoles disappear from the presumptive lens ectoderm
and the nuclei become gradually displaced toward the base of the cells in contact with the retinal surface (McKeehan, 1951). At the 16-19-somite stage the
cells become more and more columnar (so-called palisading phenomenon) and
the nuclei elongated perpendicularly to the basement membrane (lens placode
formation). According to Weiss (1947) this nuclear orientation suggests molecular orientation (perhaps as a result of the fixation of a protein molecule from
the optic vesicle to the basal surface of the presumptive lens ectoderm).
After the lens placode has been formed, its invagination begins (20-22-somite
stage, 44-50 hours of incubation) and leads gradually to the formation of a lens
vesicle, which is separated from the ectoderm at the 31-34-somite stage (63-72
hours of incubation). Soon thereafter the cells in the posterior wall of the lens
vesicle become elongated and form the first lens fibres (34-41-somite stage, 7296 hours of incubation). At the end of the 5th day a group of tall cylindrical cells
found at the equator of the lens turn 130 degrees and form new lens fibres, which
join peripherally the central fibres. This marginal zone formation reaches completion at the 8th day.
To determine at what stage of morphological development chemical substances characteristic for the adult lens appear, Burke et al. (1944) applied the
1
This work was supported by grants from the National Cancer Institute of Canada and the
National Research Council of Canada.
2
Author's address: Department of Anatomy, Medical Building, McGill University, Montreal,
Canada.
[J. Embryol. exp. Morph. Vol. 7, Part 2, pp. 193-202, June 1959]
194 J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS
complement-fixation technique and were able to detect the presence of lens antigens in 146-hour embryos, that is, at a time when the first marginal lens fibres
have been formed, but not earlier. Ten Cate & Van Doorenmaalen (1950), applying microprecipitin tests, found lens antigens as early as the 30-somite stage,
when the lens vesicle is still open to the surface. Langman, Schalekamp, Kuijken, & Veen (1957) searched for lens antigens in chick embryos from 7 to 20
somites by explanting presumptive lens ectoderm with the associated eye-cup
into a medium which contained lens antibodies. Degeneration of presumptive
lens ectoderm was found in explants cultured in this medium, but not in controls.
In the present work, these experiments were repeated under different experimental conditions using presumptive lens ectoderm not only with, but also without the associated eye-cup. The latter procedure made it possible to avoid the
inductive influence of the eye-cup during the explantation period and to examine
whether the first lens antigens appear before or during the contact period. In
addition, these experiments gave information on the relationship between chemical and morphological differentiation in presumptive lens ectoderm cells.
MATERIAL AND METHODS
In a first series of experiments, eye-cup material covered with presumptive
lens ectoderm obtained from White Leghorn chick embryos of 5-20 somites was
explanted in a fluid medium composed of equal parts of 8^-day chick embryo
extract, Tyrode solution, and rabbit serum containing antibodies against adult
lenses. The antiserum was prepared by injecting chicken lens extract (10 per
cent.) into healthy rabbits, using an injection schedule devised by Ten Cate &
Van Doorenmaalen (1950). In the control series the anti-lens serum was replaced
either by serum of normal healthy rabbits (normal serum) or by serum of rabbits
injected with the myosin fraction of frog muscle (DeHaan, 1954).
In the second series of experiments, presumptive lens ectoderm tissue from
7-20-somite chick embryos was dissected free from the eye-cup. The first
attempts to do so resulted in damage to the presumptive lens ectoderm due to
difficulties in separating the tightly bound ectoderm and eye-cup. Success was
achieved after dipping the heads of the embryos in trypsin solution (1:10,000)
for 1 minute and rinsing in Tyrode solution. It was then possible to dissect the
presumptive lens ectoderm without damaging the cells. The ectoderm cells were
explanted on a clot which had the same constituents as the experimental medium
mentioned above, but to which an equal amount of chicken plasma was added
in order to obtain a coagulum. In the control series the anti-lens serum was
replaced by 'normal' serum.
The titre of the lens antiserum used in these experiments was determined by
Boyd's method and found to vary from 1:6,400 to 1:16,000. To determine the
specificity of the lens antiserum it was tested with extracts from a variety of
organs and tissues of adult chickens. No precipitin reaction was found with most
J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS 195
organs and tissues used, except with iris, retina, cornea, and vitreous body (Langman etal, 1957).
RESULTS
Presumptive lens ectoderm with associated eye-cup cultured in 'normal' and
'anti-myosin' medium for 72 hours gave rise to lens formation in most cases
(Text-fig. 1). The percentage of lens-forming explants was found to increase with
the age of the embryo from which the tissue is obtained. Failure of lens forma% lens
forming
explants
normal medium
.
anti-myosin medium
anti-lens medium
100 .
5-9
10-11
12-13
14-15
16-17
18-19
20-21 nO. of Somites
TEXT-FIG. 1. Percentage of lenses developing in explants of optic vesicle and presumptive lens
ectoderm in various media. T h e horizontal axis represents the age of embryo at explantation.
tion in some of the explants cultured in the control media may be due to mechanical and thermal damage during dissection and heterologous constituents in the
culture medium (Langman, 1953 a, b). Microscopic study of the explants cultured in control media showed a normal, although delayed, morphological
development of the lens in comparison with in vivo development. Furthermore,
the explants cultured in 'anti-myosin' medium showed almost no mesenchyme
between the ectodermal derivatives, or only some necrotic cellular material.
Lens, optic cup, and surface ectoderm were normal.
The explants cultured in 'anti-lens' medium for 72 hours behaved differently.
In studying these tissues during the explantation period under the dissecting
microscope, it was observed that 10-20 hours after the beginning of the tissueculture period the explants showed a white, degenerating area in the presumptive lens ectoderm. This area became larger during the next few hours and often
196 J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS
broke off from the explant. Thus in some of the cultures the uncovered optic
vesicle protruded through a collar of non-presumptive lens ectoderm, whereas
the necrotic presumptive lens area dropped to the bottom of the container (Textfig. 2). However, it may be seen from Text-fig. 1 that this pattern was not followed
by all explants. All those obtained from 5-16-somite embryos were unable to
form a lens in anti-lens medium, those from 16 to 17 somites developed a lens in
24 per cent, of the cases, while the explants from embryos of 18 and more somites
behaved like the controls and formed a lens.
10 Somite
After 10hrs. culture period
|
Pres. lens
ectoderm
D
Optic
vesicle
Pig
irSMJ
After 24hrs. culture period
™,. ,, ,
^K^rlead ectoderm
Optic
vesicle
TEXT-FIG. 2. Diagram of changes in explants of optic vesicle and presumptive lens ectoderm from
10-somite embryos cultured in 'anti-lens' medium. On the left, at explantation. Middle, degenerating presumptive lens area after 10 hours. On the right, degenerate lens area lost, optic vesicle
protruding through ectoderm.
This experiment, which exactly confirms under our present laboratory conditions a series of data previously obtained (Langman et ai, 1957), indicates that
'normal' and 'anti-myosin' media do not interfere with lens formation in vitro',
'anti-lens' medium, however, prevents lens development in explants obtained
from chick embryos of 5-17 somites. Presumably the lens antibodies present in
the culture medium are able to react with constituents of the presumptive lens
ectoderm cells in such a manner that the cells are unable to survive.
This experiment did not decisively show whether lens antigens arise during or
before the induction period (9-20 somites). It was possible that lens antigens
were not yet present in the presumptive lens ectoderm cells of a 7-somite embryo
but arose under the influence of the associated eye-cup at some time during the
explantation period. The result of a 72-hour explantation period in an 'anti-lens'
medium would then be the same, namely, degeneration of the presumptive lens
ectoderm. Presumptive lens ectoderm from 7-20-somite embryos was therefore
dissected free from the eye-cup and explanted on a clot. By doing so it was possible to exclude the inductive influence of the eye-cup during the 72-hour culture
period.
Presumptive lens ectoderm explants without associated eye-cup obtained
J. L A N G M A N - F I R S T APPEARANCE OF SPECIFIC LENS ANTIGENS
197
from embryos of 7-20 somites were studied under the microscope during the
explantation period. Twelve to twenty-four hours after the beginning of the culture period explants on 'normal' medium showed migration and outgrowth in the
form of a membrane in or on the medium. At the end of the explantation period
80-100 per cent, of the explants showed outgrowth, whatever the age of the
donors, that is to say, regardless of whether or not the ectoderm had been exposed to the inductive influence of the eye-cup prior to explantation. However,
although the cells looked healthy and migrated, no lenses were formed.
% outgrowing
explants
too ,
80
60
40
normal medium
anti-lens medium
K
\
\
\
v
A
\/
)
\
\
\
/
/
\
\
\
\
\
\
\
\
\
i
\
\
20
\
\
\
*
10 II 12 13 14 15 16 17 18 19 20 no. of somites
TEXT-FIG. 3. Percentage of explants of presumptive lens ectoderm (optic vesicle removed) showing
outgrowth after 72 hours in culture in normal or 'anti-lens' medium. The horizontal axis represents
the age of embryo at explantation.
The explants grown on 'anti-lens' medium behaved differently according to
the age of the embryos. Presumptive lens ectoderm obtained from 7-10-somite
embryos showed outgrowth as explants in normal medium. Cultures obtained
from 11-12-somite embryos showed outgrowth only in about 30 per cent, of the
cases and those from 13-16-somite embryos in none. In the latter, disintegration
and death of the cells were observed in all cases 12-24 hours after the beginning
of the culture period. Fragments obtained from embryos beyond the 17-somite
stage again showed outgrowth and migration just as the control explants did
(Text-fig. 3; Table 1).
From this experiment it can be concluded that, shortly after the 9-10-somite
stage, when direct contact between eye-cup and ectoderm is established and induction is thus elicited, the cells lose their ability to survive in 'anti-lens' medium.
5584.7
198 J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS
A new component somehow appears in the presumptive lens ectoderm cells and
reacts with the lens antibodies in the medium in such a way that the cells are
unable to survive. Toward the end of the induction period (18 somites), when
adhesion between eye-cup and ectoderm is diminishing, the presumptive lens
cells are no longer killed by lens antibodies.
TABLE 1
Influence of 'normal' and 'anti-lens' medium on
presumptive lens ectoderm
Number of
somites
1
8
9
10
11
12
13
14
15
16
17
18
19
20
Number ofexpi „.,,„ outgrowing
total
Normal medium
Anti-lens medium
e
6
u
5
6
7
7
6
7
2
8
4
12
0
11
0
15
0
20
0
80
8
20
9
12
13
16
11
13
6
7
'i
5
6"
9
Yl
8
10
12
13
11
Y'3"
9
Yl
10
11
10
11
.11
13
9
10
10
ll
.10.
11
DISCUSSION
Antibodies produced by injecting specific substances—antigens—into rabbits
have the capacity to react selectively with these antigens and with closely related
substances. This reaction, which is based upon the formation of an antibodyantigen complex, can become visible as a flocculation, agglutination, precipitate,
cell degeneration, &c. By testing extracts of unknown composition with antibodies prepared against a well-defined organ, it is possible to detect whether or
not antigens specific for this organ are present in the unknown extracts. Applying this immunological detection method in embryology, it was found that antibodies prepared with antigens of adult organs and tissues may react with antigenically active materials in embryos—a fact indicating that embryonic organs
and tissues may possess antigenic substances which are identical or closely
related to adult antigens. Some of these embryonic substances persist up to the
adult stage, while others are transitory and disappear during development
(Ebert, 1953; Tyler, 1955; Nace, 1955).
J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS 199
7-10-somite stage
By culturing presumptive lens ectoderm without the associated eye-cup on a
medium containing lens antibodies it was found that explants obtained from
7-10-somite embryos showed the same active outgrowth and migration as the
controls (Text-fig. 3). Thus at this stage of development no antigenic substances
capable of reacting with lens antibodies were detectable. Examination of presumptive lens ectoderm at this stage using the agar gel technique of Ouchterlony (1948) showed no lens antigens either (unpublished data). Although it is
known that the eye-cup has started exerting its inductive influence upon the
presumptive lens ectoderm cells at the 9-10-somite stage these cells do not yet
contain any chemicals related to lens proteins. Since there is no morphological
distinction from the surrounding head ectoderm either, it may be concluded that
no detectable chemical or morphological differentiation has yet taken place.
11-17-som ite stage
As shown in the introduction, the cells of the presumptive lens ectoderm start
morphological differentiation at the 13-somite stage. Explantation of presumptive lens ectoderm with or without the associated eye-cup from 11-17-somite
embryos in a medium containing lens antibodies produced degeneration of the
cells (Text-figs. 1 and 3). This result means that substances able to react with lens
antibodies arose in the presumptive lens ectoderm at the 11-somite stage, that is,
shortly before the cells become distinguishable from the surrounding head ectoderm cells. Hence the first lens antigens seemed to be formed shortly before the
first features of morphological differentiation appeared (13 somites).
The lens antisera used in our experiments were prepared with 'adult' lens
material. Does this mean that the antigens detected in presumptive lens cells by
use of these antisera were the same as in the adult? In testing the presence of lens
antigen in chick embryos by means of the precipitin method, Ten Cate & Van
Doorenmaalen (1950) concluded that 'adult' lens antigen was present at the 30somite stage. However, since Landsteiner (1947) found that the specificity of
antigenic structures may be represented by relatively small chemical groups on
a large molecule rather than by the molecule as a whole, the presumptive lens
cells need not necessarily have identical antigens, but merely identical chemical
groups.
The fact that morphological lens differentiation was preceded by the appearance of specific chemical units suggests that, under the inductive influence of the
eye-cup, some components of the indifferent multi-potent head ectoderm cells
acquired the determining groups characteristic of lens antigens. These antigens
appearing at the 11-somite stage (first-stage antigens) may give rise to a number
of antigenic structures, each preceding the appearance of a new morphological
structure as the placode transforms through lens vesicle to adult lens. Francois
et al. (1956) showed by immunochemical analysis of extract of adult lens
material that at least eight different antigens are present at the adult stage.
200 J. LANGMAN—FIRST APPEARANCE OF SPECIFIC LENS ANTIGENS
A possible mechanism by which antibodies kill presumptive lens ectoderm
cells during the 11-17-somite stage may be a cell surface reaction. This antigenantibody surface reaction may interfere with exchange processes between the
cell and its environment, or cause some other change in surface properties (surface effect). Another possibility is the passage of antibodies through the cell
membrane and the combination with substances within the cell (intracellular
effect). The antibody-antigen binding may block the function of a cell substance
essential for cell growth and survival, or the formed complex may be toxic and
thus kill the cell. The entry of the antibody into the cell presumed to occur
during the 11-17-somite stage may be an indication of a high permeability of
the cell membrane.
18- and more than 18-somite stage
At this stage of development, when the 'first stage' lens antigens have been
formed, the differentiation of the lens cells can proceed without any further need
for the eye-cup. This was demonstrated by McKeehan (1954), who transplanted
placode cells of a 21-somite embryo into the coelomic cavity of another chick
embryo. This transplant showed the capacity to form a small lens, indicating
independent self-differentiation at this stage of development. Similarly, it appeared from our experiments that at the 18-somite stage the future lens cells are
no longer sensitive to lens antibodies (Text-figs. 1 and 3). A possible explanation
was that the 'first stage' lens antigens were transitional antigens. This was unlikely
since, using the same antilens serum but a different technique (Ouchterlony,
1948) we demonstrated the presence of lens antigens at the 18-22-somite stage
(unpublished).
An alternative explanation is that the 'surface effect' no longer takes place,
perhaps because of a change in the cell membrane. That there are indeed
changes in the cell membrane is suggested by the fact that at the 18-somite stage
the presumptive lens cells can be separated from the eye-cup without treatment
by trypsin solution. This change in the cell membrane may also prevent the
access of antibodies into the cells and thus avoid their 'intracellular effect'. If,
however, the intracellular reaction is not blocked, it may be that the neutralized
antigens are no longer toxic to cell life or, if the original antigens were essential,
they can now be replaced by other substances.
From these experiments it may be concluded that lens antibodies applied in
tissue culture can act as sensitive indicators of the formation or presence of
specific substances (lens antigens) in embryonic cells. The formation of an
antigen-antibody complex leads to the death of the presumptive lens ectoderm
cells only during the 11-17-somite stage. Before and after this stage the cells are
not visibly affected by the presence of lens antibodies, indicating either that the
reacting antigens are not present or that the formation of an antigen-antibody
complex is prevented. The most likely mode of antibody action is considered to
J. LANGMAN—PIRST APPEARANCE OF SPECIFIC LENS ANTIGENS 201
be the 'intracellular effect', the cell membrane, however, having an important
function in admitting the antibodies into the cells.
SUMMARY
1. Presumptive lens ectoderm with associated eye-cup from 5-17-somite chick
embryos was explanted in 'normal' medium, 'anti-myosin' medium, and a
medium containing lens antibodies. The control explants in 'normal' and 'antimyosin' medium showed lens formation in 80-100 per cent, of the cases (Textfig. 1); the explants in 'anti-lens' medium showed degeneration and necrosis of
the future lens area, indicating the presence of substances (lens antigens) capable
of reacting with lens antibodies (Text-fig. 1). Explants from embryos with 18 or
more somites in 'anti-lens' medium formed lenses as the controls.
2. Presumptive lens ectoderm without the associated eye-cup from 7-20somite embryos was cultured on a 'normal' and 'anti-lens' medium. The controls showed epithelial membranous outgrowth in 80-100 per cent, of the
explants (Text-fig. 3; Table 1). Explants on 'anti-lens' medium showed outgrowth
similar to controls when obtained from embryos younger than 11 and older than
17 somites, but showed degeneration when obtained from 11- to 17-somite embryos, indicating the formation of the first reacting substances (lens antigens) at
the 11-somite stage (Text-fig. 3; Table 1).
3. The 'first-stage' lens antigens are formed at the 11-somite stage, shortly
after the adhesion between eye-cup and presumptive lens ectoderm has been
established (9 somites) and shortly before the morphological changes characterized by loss of vacuolization, nuclear orientation, and palisade phenomenon
appear (13—19 somites).
ACKNOWLEDGEMENTS
I wish to thank Dr. C. P. Leblond for his helpful and critical suggestions about
this work, and Miss R. Veen for technical assistance in the experiments.
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(Manuscript received 20: x: 58)